DNA Sequencing pipeline for 454 GS/FLX runs

This is the pipeline process for 454 sequencing. I am not a biotech guy but after spending time, giving effort and getting help from my collague, I came up with a way to do that. The tools I used are NCBI SRA Toolkit and 454 sequencing tool for multiplexing.

process

• Downloaded SRA files from …

• Converted these SRA files into SFF format using sff-dump tool

    	$ sff-dump -A xxxx.sra
    

• Rebuilt the scores of converted sff dataset with sfffile tool

    	$ sfffile -o xxxxxxn.sfff xxxxx.sff
    

• Split the file according to MID groupname

    	$ sfffile -s GSMIDs/RLMIDs xxxxx.sff
    

• Calculated the total MID matches for each group
  • Extract sequence:

		 $ sffinfo -s 454Readsxx.sff > MIDx.fasta
		

*  Count total sequence no:  		

		 $ egrep -e '^>'  MIDx.fasta | wc -l
		

• Combine sff files into one main file

	$ sfffile -o combined.sff xxx1.sff xxx2.sff xxx3.sfff ...
	

Other useful Commands

• Get the quality scores from the sff file:

	$ sffinfo -q 454Readsxx.sff > MIDx.qual
	

• Retrieve the flow intensities:

	$ sffinfo -f 454Readsxx.sff > MIDx.flow
	

• View file:

	$ more/less MIDx.fasta